Use of the covariance matrix in directly fitting kinetic parameters: application to GABAA receptors

A new method of analysis is described that begins to explore the relationship between the phases of ion channel desensitization and the underlying states of the channel. The method, referred to as covariance fitting (CVF), couples Q-matrix calculations with a maximum likelihood algorithm to fit macroscopic desensitization data directly to kinetic models. Unlike conventional sum-of-squares minimization, CVF fits both the magnitude of the recorded current and the strength of the correlations between different time points. When applied to simulated data generated using various kinetic models with up to 11 free parameters, CVF leads to reasonable parameter estimates. Coupled with the likelihood ratio test, it accurately discriminates between models with different numbers of states, discriminates between most models with the same number but a different arrangement of states, and extracts meaningful information on the relationship between the desensitized states and the phases of macroscopic desensitization. When applied to GABA(A) receptor traces (outside out patches, alpha 1 beta 2 gamma 2S, 1 mM GABA, >2.5 s), a model with two open states and three desensitized states is favored. When applied to simulated data generated using a consensus model, CVF leads to reasonable parameter estimates and accurately discriminates between this and other models.     

Cytochrome P450(cin) (CYP176A), isolation,expression, and characterization

Cytochromes P450 are members of a superfamily of hemoproteins involved in the oxidative metabolism of various physiologic and xenobiotic compounds in eukaryotes and prokaryotes. Studies on bacterial P450s, particularly those involved in monoterpene oxidation, have provided an integral contribution to our understanding of these proteins, away from the problems encountered with eukaryotic forms. We report here a novel cytochrome P450 (P450(cin), CYP176A1) purified from a strain of Citrobacter braakii that is capable of using cineole 1 as its sole source of carbon and energy. This enzyme has been purified to homogeneity and the amino acid sequences of three tryptic peptides determined. By using this information, a PCR-based cloning strategy was developed that allowed the isolation of a 4-kb DNA fragment containing the cytochrome P450(cin) gene (cinA). Sequencing revealed three open reading frames that were identified on the basis of sequence homology as a cytochrome P450, an NADPH-dependent flavodoxin/ferrodoxin reductase, and a flavodoxin. This arrangement suggests that P450(cin) may be the first isolated P450 to use a flavodoxin as its natural redox partner. Sequencing also identified the unprecedented substitution of a highly conserved, catalytically important active site threonine with an asparagine residue. The P450 gene was subcloned and heterologously expressed in Escherichia coli at approximately 2000 nmol/liter of original culture, and purification was achieved by standard protocols. Postulating the native E. coli flavodoxin/flavodoxin reductase system might mimic the natural redox partners of P450(cin), it was expressed in E. coli in the presence of cineole 1. A product was formed in vivo that was tentatively identified by gas chromatography-mass spectrometry as 2-hydroxycineole 2. Examination of P450(cin) by UV-visible spectroscopy revealed typical spectra characteristic of P450s, a high affinity for cineole 1 (K(D) = 0.7 microm), and a large spin state change of the heme iron associated with binding of cineole 1. These facts support the hypothesis that cineole 1 is the natural substrate for this enzyme and that P450(cin) catalyzes the initial monooxygenation of cineole 1 biodegradation. This constitutes the first characterization of an enzyme involved in this pathway.     

Variations in transforming growth factor beta in human milk are not related to levels in plasma

There is considerable variability in the concentrations of transforming growth factor beta (TGFbeta) in human milk from individual women which is not readily explained by maternal factors such as smoking or illness. A potential correlate is the maternal plasma level of TGFbeta since changes in plasma concentration of TGFbeta in response to a number of pathological conditions have been well documented. The purpose of this study was to investigate the relationship between the concentration of TGFbeta1 and TGFbeta2 in a single sample of human milk and plasma obtained on the same day from 80 lactating mothers at 5 weeks postpartum. The concentration of TGFbeta1 and TGFbeta2 in the aqueous fraction of human milk ranged from 228 to 3542 pg/ml (647, 438-799; median, 25th-75th percentiles) and 98 to 13 855 pg/ml (955, 535-1999) respectively and in paired samples of plasma from 440 to 19 460 (4026, 3245-6656) and 92 to 1739 (620, 391-925) respectively. Thus, in milk the median ratio of TGFbeta1 to TGFbeta2 was 1:1.6 whereas the corresponding median ratio in plasma was 7:1. There was no correlation between the concentration of either isoform of TGFbeta in milk and the corresponding TGFbeta in plasma.     

Male strategies and Plio-Pleistocene archaeology

Archaeological data are frequently cited in support of the idea that big game hunting drove the evolution of early Homo, mainly through its role in offspring provisioning. This argument has been disputed on two grounds: (1) ethnographic observations on modern foragers show that although hunting may contribute a large fraction of the overall diet, it is an unreliable day-to-day food source, pursued more for status than subsistence; (2) archaeological evidence from the Plio-Pleistocene, coincident with the emergence of Homo can be read to reflect low-yield scavenging, not hunting. Our review of the archaeology yields results consistent with these critiques: (1) early humans acquired large-bodied ungulates primarily by aggressive scavenging, not hunting; (2) meat was consumed at or near the point of acquisition, not at home bases, as the hunting hypothesis requires; (3) carcasses were taken at highly variable rates and in varying degrees of completeness, making meat from big game an even less reliable food source than it is among modern foragers. Collectively, Plio-Pleistocene site location and assemblage composition are consistent with the hypothesis that large carcasses were taken not for purposes of provisioning, but in the context of competitive male displays. Even if meat were acquired more reliably than the archaeology indicates, its consumption cannot account for the significant changes in life history now seen to distinguish early humans from ancestral australopiths. The coincidence between the earliest dates for Homo ergaster and an increase in the archaeological visibility of meat eating that many find so provocative instead reflects: (1) changes in the structure of the environment that concentrated scavenging opportunities in space, making evidence of their pursuit more obvious to archaeologists; (2) H. ergaster's larger body size (itself a consequence of other factors), which improved its ability at interference competition.     

The use of fluorescamine as a probe for labeling the outer surface of the plasma membrane

A rapid method was developed to label the outer surface of chick embryo fibroblasts with fluorescamine without disruption of the cell monolayer. Polyacrylamide gel electrophoresis resolved two distinct areas of fluorescence: a group of high molecular weight polypeptides and several rapidly migrating species. The latter were demonstrated by tlc to be phospholipids. Fluorescamine did not label internal components of the cell as evidenced by two intracellular proteins which were found to be non-fluorescent. Intact normal cells were labeled 3-fold more than transformed cells, indicating a possible loss of exposed sites at the surface, while disrupted cells, subsequently labeled, yielded similar amounts of fluorescence.     

In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold

In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin.     

Porphyra lilliputiana sp. nov. (Bangiales, Rhodophyta): A diminutive New Zealand endemic with novel reproductive biology

A new species of Porphyra, Porphyra lilliputiana, is described for the New Zealand region. This species is very small ([5] 10–20 [35] mm) and is found growing epiphytically, epilithically and epizoically on upper inter-tidal shores of moderate exposure. Field-collected material of P. lilliputiana possessed archeosporangia, endosporangia, spermatangia and zygotosporangia. In culture, archeospores vi/ere released and germinated to form thalli. Endosporangia either developed directly into thalli or released endospores which individually formed thalli. Zygotospores developed into the concho-celis phase, which formed conchosporangia. Released conchospores formed thalli. This species is distinguished by its small size, arrangement of reproductive cells, occurrence of endosporangia, dentate margin and habitat.     

Rhizobium (Sinorhizobium) meliloti phn Genes: Characterization and Identification of Their Protein Products

In Escherichia coli, the phn operon encodes proteins responsible for the uptake and breakdown of phosphonates. The C-P (carbon-phosphorus) lyase enzyme encoded by this operon which catalyzes the cleavage of C-P bonds in phosphonates has been recalcitrant to biochemical characterization. To advance the understanding of this enzyme, we have cloned DNA from Rhizobium (Sinorhizobium) meliloti that contains homologues of the E. coli phnG, -H, -I, -J, and -K genes. We demonstrated by insertional mutagenesis that the operon from which this DNA is derived encodes the R. meliloti C-P lyase. Furthermore, the phenotype of this phn mutant shows that the C-P lyase has a broad substrate specificity and that the organism has another enzyme that degrades aminoethylphosphonate. A comparison of the R. meliloti and E. coli phn genes and their predicted products gave new information about C-P lyase. The putative R. meliloti PhnG, PhnH, and PhnK proteins were overexpressed and used to make polyclonal antibodies. Proteins of the correct molecular weight that react with these antibodies are expressed by R. meliloti grown with phosphonates as sole phosphorus sources. This is the first in vivo demonstration of the existence of these hitherto hypothetical Phn proteins.     

Female subsistence strategies among Ache hunter-gatherers of Eastern Paraguay

Anthropologists have frequently proposed that sexual division of labor is produced by childcare constraints on women's subsistence work. We present data on the forest activities of Ache women that show that differences in parental investment partially account for variation in food acquisition among individual women. Data also suggest that childcare constraints are important in understanding the sexual division of labor.